Alarm related peptides and nucleic acids and diagnosis using them

ABSTRACT

We have identified a novel protein, named ALARM or δ-catenin, on the basis of its ability to bind to presenilin 1. ALARM contains 4 copies of the arm repeat and is expressed almost exclusively in brain tissue.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional (and claims the benefit of priorityunder 35 USC 120) of U.S. application Ser. No. 08/982,785, filed Dec. 2,1997, now U.S. Pat. No. 6,258,929 which claims priority from U.S.Provisional Application Serial No. 60/031,556, filed Dec.2, 1996. Thedisclosures of the prior applications are considered part of (and areincorporated by reference in) the disclosure of this application.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under AG06601 awarded bythe National Institutes of Health. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The invention is in the general field of proteins involved inAlzheimer's disease.

Various genes and gene products involved in the development ofAlzheimer's disease have been identified. Neuritic plaquescharacteristic of the disease are composed of β-amyloid (Aβ), which areoligopeptides of about 40-43 amino acids in length derived from theβ-amyloid precursor protein (βAPP). Mutations in the gene encoding βAPPare associated with some cases of familial Alzheimer's disease. Othercases of familial Alzheimer's disease have been associated withmutations in two other loci, presenilin-1 and presinilin-2.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a heretofore undescribedprotein, which has been named ALARM or δ-catenin, on the basis of itsinteraction with presenilin 1. ALARM shows a striking sequencesimilarity to members of the armadillo (arm)-plakoglobin-β cateninprotein family. In addition, ALARM transcripts are confined almostexclusively to brain tissue.

In addition to the specific human ALARM sequences provided (orcross-referenced) herein, molecules relevant to the invention includefragments of those sequences and related polypeptides, non-peptidemimetics, and nucleic acid sequences. The invention also includesantibodies to ALARM polypeptides. These polypeptides, as well as nucleicacid encoding them, can be used for a variety of diagnostic andtherapeutic applications.

In one aspect the invention features a substantially pure vertebrateALARM polypeptide, e.g, an ALARM polypeptide from a mammal such as thehuman ALARM polypeptide shown in FIG. 1 (SEQ ID NO:2).

By “protein” and “polypeptide” is meant any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation).

Polypeptides include, but are not limited to: recombinant polypeptides,natural polypeptides, and synthetic polypeptides as well as polypeptideswhich are preproteins or proproteins.

One way to ascertain purity of a preparation is by per cent dry weight.Generally, useful preparations are at least 60% by weight (dry weight)the compound of interest, i.e., an ALARM polypeptide. Preferably thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Purity canbe measured by any appropriate standard method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A“mature human ALARM” is the amino acid sequence shown in FIG. 1 SEQ IDNO:2.

Polypeptides substantially identical to mature human ALARM may have anamino acid sequence which is at least 85%, preferably 90%, and mostpreferably 95% or even 99% identical to the amino acid sequence of theALARM polypeptide of the FIG. 1. SEQ ID NO:2. When assessing sequenceidentity of polypeptides, the length of the reference polypeptidesequence will generally be at least 16 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least 50nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705).

In the case of polypeptide sequences which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

Polypeptides corresponding to one or more domains of ALARM are alsowithin the scope of the invention. Thus, also featured is a polypeptideincluding at least one antigenic determinant of ALARM, a polypeptidecomprising at least one copy of the 42 amino acid arm repeat in theALARM polypeptide, or a polypeptide comprising a βAPP binding domain ofALARM. Preferred polypeptides are those which are soluble under normalphysiological conditions.

The polypeptides of the invention can be expressed fused to anotherpolypeptide, e.g., a marker polypeptide or fusion partner. For example,the polypeptide can be fused to a hexa-histidine tag to facilitatepurification of bacterially expressed protein or a hemagglutinin tag tofacilitate purification of protein expressed in eukaryotic cells.

In another aspect, the invention also features a substantially purepolypeptide which includes a first portion and a second portion; thefirst portion includes an ALARM polypeptide and the said second portionincludes a detectable marker. The first portion can be either afull-length form of ALARM or one or more domains thereof. The firstportion is fused to an unrelated protein or polypeptide (i.e., a fusionpartner) to create a fusion protein.

The invention also includes a pharmaceutical composition which includesan ALARM polypeptide.

In still another aspect the invention features a recombinant nucleicacid encoding an ALARM polypeptide. In one preferred embodiments thenucleic acid encodes a soluble ALARM polypeptide.

The invention also features isolated nucleic acids encoding polypeptidescorresponding to one or more domains of ALARM or ALARM-relatedpolypeptides discussed above. ALARM-encoding nucleotides can include thenucleic acids shown in FIG. 1 SEQ ID NO:1, e.g., nucleotides 366-2636 ofFIG. 1 SEQ ID NO:1. Also encompassed within the invention are nucleicacid sequences that encode forms of ALARM in which sequences are alteredor deleted.

By “isolated nucleic acid” is meant nucleic acid that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived.Thus, a recombinant nucleic acid could include some or all of the 5′non-coding (e.,g., promoter) sequences which are immediately contiguousto the coding sequence. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus, such as a retrovirus; or intothe genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequence.

Nucleic acid sequences substantially identical to human ALARM sequenceshave a nucleotide sequence which is at least 85%, preferably 90%, andmost preferably 95% or even 99% identical to the amino acid sequence ofthe ALARM polypeptide of FIG. 1 SEQ ID NO:2. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 110 nucleotides.

Also within the invention are nucleic acids encoding hybrid proteins inwhich a portion of ALARM or a portion (e.g., one or more domains)thereof is fused to an unrelated protein or polypeptide (i.e., a fusionpartner) to create a fusion protein.

The nucleic acid can be isolated either as a matter of purity or byincluding in it in DNA that is a non-naturally occurring molecule; forexample, the DNA is not immediately contiguous with both of thesequences with which it is immediately contiguous (one on the 5′ end andone on the 3′ end) in the naturally occurring genome of the organismfrom which it is derived. Thus, a recombinant nucleic acid could includesome or all of the 5′ non-coding (e.,g., promoter) sequences which areimmediately contiguous to the coding sequence. Other examples are arecombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., aCDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

The nucleic acids of the invention include nucleic acids encoding ALARMpolypeptides fused to a polypeptide which facilitates secretion, e.g., asecretory sequence. Such a fused protein is typically referred to as apreprotein. The secretory sequence can be removed by the host cell toform the mature protein. Also within the invention are nucleic acidsthat encode mature ALARM fused to a polypeptide sequence to produce aninactive preprotein. Preproteins can be converted into the active formof the protein by removal of the inactivating sequence.

The invention also encompasses nucleic acids that hybridize understringent conditions to a nucleic acid encoding an ALARM polypeptide.“Stringent conditions” means hybridization at 50° C. in Church buffer(7% SDS, 0.5% NaHPO₄, 1 mM EDTA, 1% BSA) and washing at 50° C. in 2×SSC.The hybridizing portion of the hybridizing nucleic acids are preferably20, 30, 50, or 70 bases long. Preferably, the hybridizing portion of thehybridizing nucleic acid is 95% or even 98% identical to the sequence ofa portion of a nucleic acid encoding an ALARM polypeptide. Hybridizingnucleic acids of the type described above can be used as a cloningprobe, a primer (e.g., a PCR primer), or a diagnostic probe. Preferredhybridizing nucleic acids encode a polypeptide having some or all of thebiological activities possessed by naturally-occurring ALARM.Hybridizing nucleic acids can be splice variants encoded by one of theALARM genes described herein. Thus, they may encode a protein which isshorter or longer than the various forms of ALARM described herein.Hybridizing nucleic acids may also encode proteins which are related toALARM (e.g, proteins encoded by genes which include a portion having arelatively high degree of identity to an ALARM gene described herein).

The term “nucleic acid” encompasses both RNA and DNA, including cDNA,genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Thenucleic acid may be double-stranded or single-stranded. Wheresingle-stranded, the nucleic acid may be the sense strand or theantisense strand.

In yet another aspect, the invention features vectors which include anucleic acid of the invention. In one preferred embodiment, the nucleicacid of the invention is properly positioned for expression.

By “positioned for expression” is meant that the selected DNA moleculeis positioned adjacent to one or more sequence elements which directtranscription and/or translation of the sequence such that the sequenceelements can control transcription and/or translation of the selectedDNA (i.e., the selected DNA is operably associated with the sequenceelements). Such operably associated elements can be used to facilitatethe production of an ALARM polypeptide.

In a still further aspect, the invention features transformed cellsharboring a nucleic acid encoding ALARM sequences discussed above.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding (as used herein) ALARM polypeptide.

The invention also features purified antibodies which specifically bindan ALARM protein or polypeptide.

By “purified antibody” is meant an antibody which is at least 60%, bydry weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, antibody.

By “specifically binds” is meant an antibody that recognizes and bindsto and forms a complex with, a particular antigen, e.g., ALARMpolypeptide, but which does not substantially recognize and bind toother molecules in a sample, e.g., a biological sample, which naturallyincludes ALARM.

The invention also features a method of diagnosing in a mammal, e.g., ahuman subject, an increased likelihood of, inclination toward, orsusceptibility to developing a disease, in which a mutant form of theALARM protein is a causative agent. The same method is also used todiagnose the ability of a mammal, e.g., a human, to transmit to futuregenerations a mutant form of a protein which is a causative agent of adisease. The method involves analyzing the DNA of the mammal todetermine the presence or absence of a mutation in a gene for an ALARMprotein, the presence of such a mutation indicating the increasedlikelihood. Preferably the DNA is analyzed by amplifying the DNA with,e.g., the polymerase chain reaction, and identifying mutations in theDNA by use of the single-strand conformation polymorphism (SSCP)technique, as used and described herein, or by direct DNA sequencing.

In another aspect, the invention includes a method of inhibitingexpression of an ALARM gene comprising administering to a cellcontaining an ALARM transcript an anti-sense ALARM oligonucleotide.

The invention also includes a method of detecting presenilin 1 in asample, e.g., a sample taken from a human, comprising contacting thesample with an ALARM polypeptide. The sample can be from, e.g.,cerebrospinal fluid.

In another aspect, the invention includes a method of diagnosing in ahuman subject a disease in which a mutant form of a protein whichinteracts with ALARM is a causative agent. The method includes analyzinga sample of fluid from the human subject to determine the presence orabsence of the ALARM-interacting protein.

The invention further includes a method of diagnosing in a human subjectan increased likelihood of developing or transmitting to futuregenerations a disease in which a mutant form of a human ALARM is acausative agent. The method includes analyzing the DNA of the subject todetermine the presence or absence of a mutation in a gene for an ALARMprotein, the presence of such a mutation indicating the increasedlikelihood of transmitting the disease. The method can include, e.g.,amplifying the DNA of the subject, DNA sequencing, or identifying asingle strand conformation polymerism.

The invention also includes a probe or primer comprising a substantiallypurified single-stranded oligonucleotide, e.g., a DNA oligonucleotide,wherein the oligonucleotide contains a region which is identical to thesequence of a six-nucleotide, single-stranded segment of a gene encodinga mutant form of a human ALARM, wherein the segment comprising part orall of the mutation.

In yet another aspect, the invention includes a method of detecting anALARM-containing complex in a biological sample by contacting the samplewith an ALARM protein or an ALARM antibody and determining whether theALARM protein or antibody binds to a component of the sample.

In a further aspect, the invention includes a method of diagnosingaltered levels, e.g., lower or altered levels, of presenilin 1 in asample by contacting the sample with ALARM and determining whether thesample contains presenilin 1 that binds to ALARM.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed descriptions, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E are a schematic representation of the predicted nucleotideSEQ ID NO:1 and amino acid SEQ ID NO:2 sequence of the human ALARMprotein.

FIG. 2 is a schematic representation of the ALARM arm repeats SEQ IDNOS:4-7 and their homology to the Drosophila arm sequence SEQ ID NO:3.

FIG. 3 is a.,schematic representation of the ALARM SEQ ID NO:8 andpp120SEQ ID NO:9 amino acid sequences.

FIG. 4 is a schematic representation of the ALARM SEQ ID NO:10 and γcatenin SEQ ID NO:11 amino acid sequences.

DETAILED DESCRIPTION

Previously described genes encoding proteins involved in Alzheimer'sdisease include βAPP, which was isolated as the cellular protein givingrise to the polypeptide fragments found in the Aβ plaques characteristicof Alzheimer's disease (reviewed in Selkoe, Ann. Rev. Cell Biol. 10:373,1994), as well as presenilin 1 and presenilin 2, which were identifiedas cellular genes altered in cases of familial Alzheimer's disease(Sherrington et al., Nature 30 375:754, 1995; Levy-Lahad et al., Science259:970, 1995; Rogaev et al., Nature 376:207, 1995).

βAPP, presenilin-1, and presenilin-2 all encode transmembrane proteins.The protein encoded by βAPP has a type I single transmembrane segment(Selkoe, supra), while the presenilin 1 and presenilin 2 polypeptideshave seven putative transmembrane segments (Sherrington et al., supra,1995; Levy-Lahad et al., Science 269:973, 1995; Rogaev et al., supra).In addition, presenilin 1 and 2 are homologous to the sel-12 gene in thenematode, C. elegans, which likewise encodes a protein with sevenputative transmembrane segments (Leviatan et al., Nature 377:351, 1995;Grant et al., Genetics 143:237, 1996). The sel-12 gene was identified asa suppressor of defects in the lin-12 locus, which encodes a type Itransmembrane protein (Sundaram et al., Genetics 135:765, 1993; Yochemet al. Nature 335:547, 1988). Based in part on this similarity, a modelhas been proposed in which the βAPP protein binds to the presenilin-1 orpresenilin 2 gene product (Dewji et al., Science 271:159, 1996).

Until the present discovery, however, little was known about howproducts of the presenilin 1 and presenilin 2 genes interacted with eachother, with other proteins, or whether they participated in any knownsignal transduction pathways. We have used the two-hybrid yeast systemto identify a novel human protein on the basis of its interaction withthe single hydrophilic loop region of presenilin 1. The interactingprotein contains multiple copies of an amino acid repeat sequence firstdescribed in the armadillo (arm) gene in the fruit fly, Drosophilamelanogaster (Riggleman et al., Genes Develop. 3:96, 1989). Proteinswith the arm repeat have been subsequently identified in'several otherproteins, including plakoglobin, β-catenin, and p120 (Peifer et al., J.Cell Biol. 118:681, 1992); Reynolds et al., Oncogene 7:2439, 1992). Asother members of this family have been localized to the adherensjunction, the new protein has been named ALARM, for adherens-junctionlinked arm protein. Alternatively, it can also be called δ-catenin,since it shows homology to known members of the catenin protein family.

Two functions have previously been ascribed to members of the armfamily. First, evidence from diverse organisms suggests that arm isinvolved in the Wnt signal transduction pathway. Wnt homologs in avariety of organisms have been associated with signalling functionsduring animal development. In general, Wnt functions act so that groupsof cells maintain the same identity as neighboring cells. Thus, inDrosophila the Wnt homolog, wingless (wg), acts to maintain engrailedexpression in adjacent group of cells. (DiNardo et al., Nature 332:604,1988; Martinez-Aria et al., Development 103:157, 1988).

Similarly, addition of wg, to Drosophila embryos increases the level ofarm protein (Riggleman et al., Cell 63:549, 1990). This interaction ismediated through the binding of wg to cell-surface receptors encoded bymembers of the frizzled (Dfz) gene family (Bhanot et al., Nature382:225, 1996). Other Drosophila genes involved in the Wng signallingpathway include dishevelled (dsh) and zeste-white 3 (zw3) (see Bhanot etal., supra).

In Xenopus laevis embryos, ectopic expression of β-catenin results in aphenotype similar to that caused by mutations in some member of the Wntfamily (Guger et al., Dev. Biol 172:115-25). In mammalian cells, Wnt-1expression results in the accumulation of β-catenin and plakoglobin(Hinck et al., J. Cell Biol. 124:729, 1994).

β catenin also forms a complex with the transcription factor LEF-1, andthis complex localizes to the nucleus. (Behrens et al., Nature 382:638,1996). Thus, a combination of genetic and biochemical studies suggestarm family members may be involved in transducing signals from thecell-surface to the nucleus in the Wnt pathway.

The second function in which members of the arm family have beenimplicated is promotion of cell adhesion. Plakoglobin, β catenin, andp120 all associate with the cytoplasmic domains of the calcium-dependentcell-cell adhesion proteins called cadherins (Daniel et al., Mol Cell.Biol. 15:4819, 1995); Shibamoto et al., J. Cell. Biol. 128:949, 1995).pl20 is thought to associate with E-cadherin via E-cadherin's carboxylterminus (Shibamoto et al., supra). Similarly, arm proteins have beenlocalized to the cytoplasmic surface of cells and colocalize with actin.(Riggleman et al., Cell 63:549, 1990).

The present invention for the first time suggests members of the armfamily are involved in the pathology of Alzheimer's disease.

ALARM Polypeptides, Proteins and Nucleic Acid Sequences

The invention encompasses, but is not limited to, ALARM proteins andpolypeptides that are functionally related to ALARM encoded by thenucleotide sequence of FIG. 1 SEQ ID NO:1. Functionally related proteinsand polypeptides include any protein or polypeptide sharing a functionalcharacteristic with ALARM, e.g., the ability to bind to presenilin 1.Such functionally related ALARM polypeptides include, but are notlimited to, additions or substitutions of amino acid residues within theamino acid sequence encoded by the ALARM sequences described hereinwhich result in a silent change, thus producing a functionallyequivalent gene product. Amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, cryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

ALARM polypeptides and proteins of the invention can be made by alteringnucleic acid sequences encoding ALARM polypeptides. While randommutations can be made to ALARM DNA (using random mutagenesis techniqueswell known to those skilled in the art) and the resulting mutant ALARMproteins can be tested for activity, site-directed mutations of theALARM coding sequence can be engineered (using site-directed mutagenesistechniques well known to those skilled in the art) to generate mutantALARMs.

To design variant ALARM polypeptides which may be altered in theirfunction, e.g., in their ability to bind to presenilin 1, it is usefulto distinguish between conserved positions and variable positions.Conserved positions are those in which the amino acid in an ALARMprotein from another organism as in the same position as it is in thehuman ALARM protein.

To preserve ALARM function, it is preferable that conserved residues arenot altered. Moreover, alteration of non-conserved residues arepreferably conservative alterations, e.g., a basic amino acid isreplaced by a different basic amino acid. To produce altered functionvariants, it is preferable to make non-conservative changes at variableand/or conserved positions. Deletions at conserved and variablepositions can also be used to create altered function variants.

Other mutations to the ALARM coding sequence can be made to generateALARMs that are better suited for expression, e.g., scaled upexpression, in a selected host cell. For example, potential N-linkedglycosylation sites can be altered or eliminated to achieve, forexample, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionsof any one or more of the glycosylation recognition sequences whichoccur (in N-X-S or N-X-T), and/or an amino acid deletion at the secondposition of any one or more of such recognition sequences, will preventglycosylation at the modified tripeptide sequence. (See, e.g., Miyajimaet al., Embo J. 5:1193, 1986).

Preferred ALARM polypeptides are those polypeptides, or variantsthereof, which bind to presenilin 1 polypeptides. In determining whethera particular ALARM polypeptide or variant thereof binds to presenilin 1,one can use any assay techniques disclosed herein or in referencedpublications. Preferred ALARM polypeptides and variants have 20%, 40%,50%, 75%, 80%, or even 90% of the activity of the full-length, maturehuman form of ALARM described herein. Such comparisons are generallybased on equal concentrations of the molecules being compared. Thecomparison can also be based on the amount of protein or polypeptiderequired to reach 50% of the maximal stimulation obtainable.

Also within the invention are fusion proteins in which a portion (e.g.,one or more domains) of ALARM is fused to an unrelated protein orpolypeptide (i.e., a fusion partner) to create a fusion protein. Thefusion partner can be a moiety selected to facilitate purification,detection, or solubilization, or to provide some other function. Fusionproteins are generally produced by expressing a hybrid gene in which anucleotide sequence encoding all or a portion of ALARM is joinedin-frame to a nucleotide sequence encoding the fusion partner.

In general, ALARM proteins according to the invention can be produced bytransformation (transfection, transduction, or infection) of a host cellwith all or part of an ALARM-encoding DNA fragment (e.g., the ALARM DNAdescribed herein) in a suitable expression vehicle. Suitable expressionvehicles include: plasmids, viral particles, and phage. For insectcells, baculovirus expression vectors are suitable. The entireexpression vehicle, or a part thereof, can be integrated into the hostcell genome. In some circumstances, it is desirable to employ aninducible expression vector, e.g., the LACSWITCH™ Inducible ExpressionSystem (Stratagene; LaJolla, Calif.).

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems can be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. The ALARM protein can be produced in a prokaryotic host(e.g., E. coli or B. subtilis) or in a eukaryotic host (e.g.,Saccharomyces or Pichia; mammalian cells, e.g., COS, NIH 3T3 CHO, BHK,293, or HeLa cells; or insect cells).

Proteins and polypeptides can also be produced by plant cells. For plantcells viral expression vectors (e.g., cauliflower mosaic virus andtobacco mosaic virus) and plasmid expression vectors (e.g., Ti plasmid)are suitable. Such cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockland, Md. ; also, see,e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1994). The methods of transformation or transfectionand the choice of expression vehicle will depend on the host systemselected. Transformation and transfection methods are described, e.g.,in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1994); expression vehicles may be chosen from thoseprovided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwelset al., 1985, Supp. 1987).

The host cells harboring the expression vehicle can be cultured inconventional nutrient media adapted as need for activation of a chosengene, repression of a chosen gene, selection of transformants, oramplification of a chosen gene.

One preferred expression system is the mouse 3T3 fibroblast host celltransfected with a pMAMneo expression vector (Clontech, Palo Alto,Calif.). pMAMneo provides an RSV-LTR enhancer linked to adexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replicationwhich allows replication in mammalian systems, a selectable neomycingene, and SV40 splicing and polyadenylation sites. DNA encoding an ALARMprotein would be inserted into the pMAMneo vector in an orientationdesigned to allow expression. The recombinant ALARM protein would beisolated as described below. Other preferable host cells that can beused in conjunction with the pMAMneo expression vehicle include COScells and CHO cells (ATCC Accession Nos. CRL 1650 and CCL 61,respectively).

ALARM polypeptides can be produced as fusion proteins. For example, theexpression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983), can beused to create lacZ fusion proteins. The pGEX vectors can be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan be easily purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect cell expression system, Autographa californica nuclearpolyhidrosis virus (AcNPV), which grows in Spodoptera frugiperda cells,is used as a vector to express foreign genes. An ALARM coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter, e.g., the polyhedrin promoter. Successful insertion of a geneencoding an ALARM polypeptide or protein will result in inactivation ofthe polyhedrin gene and production of non-occluded recombinant virus(i.e., virus lacking the proteinaceous coat encoded by the polyhedringene). These recombinant viruses are then used to infect spodopterafrugiperda cells in which the inserted gene is expressed (see, e.g.,Smith et al., J. Virol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the ALARM nucleic acid sequence can be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene can then be inserted intothe adenovirus genome by in vitro or in vivo recombination. Insertioninto a non-essential region of the viral genome (e.g., region E1 or E3)will result in a recombinant virus that is viable and capable ofexpressing an ALARM gene product in infected hosts (see, e.g., Logan,Proc. Natl. Acad. Sci. USA 81:3655, 1984.).

Specific initiation signals may also be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire native ALARM gene or ALARM cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. In other cases, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators (Bittner etal., Methods in Enzymol. 153:516, 1987).

In addition, a host cell may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and inparticular, choroid plexus cell lines.

Alternatively, an ALARM protein can be produced by a stably-transfectedmammalian cell line. A number of vectors suitable for stabletransfection of mammalian cells are available to the public, see, e.g.,Pouwels et al. (supra); methods for constructing such cell lines arealso publicly available, e.g., in Ausubel et al. (supra). In oneexample, cDNA encoding the ALARM protein is cloned into an expressionvector that includes the dihydrofolate reductase (DHFR) gene.Integration of the plasmid and, therefore, the ALARM protein-encodinggene into the host cell chromosome is selected for by including 0.01-300μM methotrexate in the cell culture medium (as described in Ausubel etal., supra). This dominant selection can be accomplished in most celltypes.

Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are described in Ausubel et al. (supra);such methods generally involve extended culture in medium containinggradually increasing levels of methotrexate. DHFR-containing expressionvectors commonly used for this purpose include pCVSEII-DHFR andpAdD26SV(A) (described in Ausubel et al., supra). Any of the host cellsdescribed above or, preferably, a DHFR-deficient CHO cell line (e.g.,CHO DHFR-cells, ATCC Accession No. CRL 9096) are among the host cellspreferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

A number of other selection systems can be used, including but notlimited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyl-transferase, and adeninephosphoribosyltransferase genes can be employed in tk, hgprt, or aprtcells, respectively. In addition, gpt, which confers resistance tomycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147, 1981),can be used.

Alternatively, any fusion protein can be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described in Janknecht et al., Proc. Natl. Acad. Sci.USA, 88:8972, 1991), allows for the ready purification of non-denaturedfusion proteins expressed in human cell lines. In this system, the geneof interest is subcloned into a vaccinia recombination plasmid such thatthe gene's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Alternatively, ALARM or a portion thereof, can be fused to animmunoglobulin Fc domain. Such a fusion protein can be readily purifiedusing an affinity column.

ALARM proteins and polypeptides can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees, can be used togenerate ALARM-expressing transgenic animals.

Any technique known in the art can be used to introduce an ALARMtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (U.S. Pat. No. 4,873,191); retrovirus mediated genetransfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci.,USA 82:6148, 1985); gene targeting into embryonic stem cells (Thompsonet al., Cell 56:313, 1989); and electroporation of embryos (Lo, Mol.Cell. Biol. 3:1803, 1983).

The present invention provides for transgenic animals that carry theALARM transgene in all their cells, as well as animals that carry thetransgene in some, but not all of their cells, i.e., mosaic animals. Thetransgene can be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems The transgene canalso be selectively introduced into and activated in a particular celltype (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232, 1992). Theregulatory sequences required for such a cell-type specific activationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

When it is desired that the ALARM transgene be integrated into thechromosomal site of the endogenous ALARM gene, gene targeting ispreferred. Briefly, when such a technique is to be used, vectorscontaining some nucleotide sequences homologous to an endogenous ALARMgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene also can be selectively introduced into a particular celltype, thus inactivating the endogenous ALARM gene in only that cell type(Gu et al., Science 265:103, 1984). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art.

Once transgenic animals have been generated, the expression of therecombinant ALARM gene can be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of ALARM gene-expressing tissue, also canbe evaluated immunocytochemically using antibodies specific for theALARM transgene product.

Once the recombinant ALARM protein is expressed, it is isolated.Secreted forms can be isolated from the culture media, whilenon-secreted forms must be isolated from the host cells. Proteins can beisolated by affinity chromatography. In one example, an anti-ALARMprotein antibody (e.g., produced as described herein) is attached to acolumn and used to isolate the ALARM protein. Lysis and fractionation ofALARM protein-harboring cells prior to affinity chromatography can beperformed by standard methods (see, e.g., Ausubel et al., supra).Alternatively, an ALARM fusion protein, for example, an ALARM-maltosebinding protein, an ALARM-β-galactosidase, or an ALARM-trpE fusionprotein, can be constructed and used for ALARM protein isolation (see,e.g., Ausubel et al., supra; New England Biolabs, Beverly, Mass.).

Once isolated, the recombinant protein can, if desired, be furtherpurified, e.g., by high performance liquid chromatography using standardtechniques (see, e.g., Fisher, Laboratory Techniques In Biochemistry AndMolecular Biology, eds., Work and Burdon, Elsevier, 1980).

Polypeptides of the invention, particularly short ALARM fragments, canalso be produced by chemical synthesis (e.g., by the methods describedin Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,Rockford, Ill.).

These general techniques of polypeptide expression and purification canalso be used to produce and isolate useful ALARM fragments or analogs(described herein).

The invention also features proteins which interact with ALARM and areinvolved in the function of ALARM. Also included in the invention arethe genes encoding these interacting proteins. Interacting proteins canbe identified using methods known to those skilled in the art. Onemethod suitable method is the “two-hybrid system,” detects proteininteractions in vivo (Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578,1991). A kit for practicing this method is available from Clontech (PaloAlto, Calif.).

Anti-ALARM Antibodies

Human ALARM proteins and polypeptides (or immunogenic fragments oranalogs) can be used to raise antibodies useful in the invention; suchpolypeptides can be produced by recombinant or peptide synthetictechniques (see, e.g., Solid Phase Peptide Synthesis, supra; Ausubel etal., supra). In general, the peptides can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Antibodies can be purifiedby peptide antigen affinity chromatography.

In particular, various host animals can be immunized by injection withan ALARM protein or polypeptide. Host animals include rabbits, mice,guinea pigs, and rats. Various adjuvants can be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Antibodies within the invention include monoclonal antibodies,polyclonal antibodies, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, and molecules producedusing a Fab expression library.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the ALARM proteinsdescribed above and standard hybridoma technology (see, e.g., Kohler etal., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976;Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981;Ausubel et al., supra).

In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described in Kohler et al., Nature256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the Mab of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this the presently preferred method of production.

Once produced, polyclonal or monoclonal antibodies are tested forspecific ALARM recognition by Western blot or immunoprecipitationanalysis by standard methods, e.g., as described in Ausubel et al.,supra.

Preferably, antibodies of the invention are produced using fragments ofthe ALARM protein which lie outside highly conserved regions and appearlikely to be antigenic, by criteria such as high frequency of chargedresidues. In one specific example, such fragments are generated bystandard techniques of PCR, and are then cloned into the PGEX expressionvector (Ausubel et al., supra). Fusion proteins are expressed in E. coliand purified using a glutathione agarose affinity matrix as described inAusubel, et al., supra.

Antisera can be raised by injections in a series, preferably includingat least three booster injections. In some cases it may be desirable tominimize the potential problems of low affinity or specificity ofantisera. In such circumstances involving fusion proteins, two or threeALARM fusion proteins can be generated for each protein, and each fusionprotein can be injected into at least two rabbits.

Antisera is also checked for its ability to immunoprecipitaterecombinant ALARM proteins or control proteins, such as glucocorticoidreceptor, CAT, or luciferase.

The antibodies can be used, for example, in the detection of the ALARMin a biological sample as part of a diagnostic assay. Antibodies alsocan be used in a screening assay to measure the effect of a candidatecompound on expression or localization of ALARM. Additionally, suchantibodies can be used in conjunction with the gene therapy techniquesdescribed to, for example, evaluate the normal and/or engineeredALARM-expressing cells prior to their introduction into the patient.Such antibodies additionally can be used in a method for inhibitingabnormal ALARM activity.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci., 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine Mab and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; and U.S. Pat. Nos. 4,946,778 and4,704,692) can be adapted to produce single chain antibodies against anALARM protein or polypeptide. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to the ALARM can, in turn, be used to generate anti-idiotypeantibodies that resemble a portion of ALARM using techniques well knownto those skilled in the art (see, e.g., Greenspan et al., FASEB J.7:437, 1993; Nissinoff, J. Immunol. 147:2429, 1991). For example,antibodies that bind to ALARM and competitively inhibit the binding of aligand of ALARM can be used to generate anti-idiotypes that resemble aligand binding domain of ALARM and, therefore, bind and neutralize aligand of ALARM. Such neutralizing anti-idiotypic antibodies or Fabfragments of such anti-idiotypic antibodies can be used in therapeuticregimens.

ALARM Oligonucleotide Diagnostic and Therapeutic Agents

Oligonucleotide therapeutic agents can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (as described, e.g., in Letsinger et al., Proc. Natl. Acad.Sci. USA 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134), orhybridization-triggered cleavage agents (see, e.g., Krol et al.,BioTechniques 6:958, 1988), or intercalating agents (see, e.g., Zon,Pharm. Res. 5:539, 1988). To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

The oligonucleotide may comprise at least one modified base moiety whichis selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

The oligonucleotide may also comprise at least one modified sugar moietyselected from the group including, but not limited to, arabinose,2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or an analog of any of thesebackbones.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids. Res. 15:6625, 1987). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett. 215:327,1987).

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209, 1988), andmethylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA85:7448, 1988).

The nucleic acid molecules should be delivered to cells that expressALARM in vivo, e.g., brain, heart, kidney, lung, uterus, endothelialcells, fibroblasts, and bone marrow stromal cells. A number of methodshave been developed for delivering DNA or RNA to cells; e.g., moleculescan be injected directly into the tissue site, or modified molecules,designed to target the desired cells (e.g., linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically.

If intracellular concentrations of the molecule sufficient to suppresstranslation of endogenous mRNAs are not immediately achieved, apreferred approach uses a recombinant DNA construct in which theoligonucleotide is placed under the control of a strong pol III or polII promoter. The use of such a construct to transfect target cells inthe patient will result in the transcription of sufficient amounts ofsingle stranded RNAs that will form complementary base pairs with theendogenous ALARM transcripts and thereby prevent translation of theALARM MRNA. For example, a vector can be introduced in vivo such that itis taken up by a cell and directs the transcription of an RNA. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired RNA.

Such vectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression it mammalian cells.Expression of the-sequence encoding the RNA can be by any promoter knownin the art to act in mammalian, preferably human cells. Such promoterscan be inducible or constitutive. Such promoters include, but are notlimited to: the SV40 early promoter region (Bernoist et al., Nature290:304, 1981); the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., Cell 22:787-797, 1988); the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA78:1441, 1981); or the regulatory sequences of the metallothionein gene(Brinster et al., Nature 296:39, 1988).

Any type of plasmid, cosmid, YAC, or viral vector can be used to preparethe recombinant DNA construct which can be introduced directly into thetissue site; e.g., the brain, kidney or heart cells. Alternatively,viral vectors can be used that selectively infect the desired tissue(e.g., for brain, herpesvirus vectors may be used), in which caseadministration can be accomplished by another route (e.g.,systemically).

Alternatively, endogenous ALARM gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the ALARM gene (i.e., the ALARM promoter and/or enhancers) toform triple helical structures that prevent transcription of the ALARMgene in target cells in the body (Helene, Anticancer Drug Des. 6:569,1981; Helene et al., Ann. N.Y. Accad. Sci. 660:27, 1992; and Maher,Bioassays 14:807, 1992).

Identification of Proteins which Interact with ALARM

The invention also features proteins which interact with ALARM. Forexample, an ALARM protein or a fusion protein containing ALARM can beused to detect the presence of presenilin 1 in a sample. Any methodsuitable for detecting protein-protein interactions may be employed foridentifying transmembrane proteins, intracellular, or extracellularproteins that interact with ALARM. Among the traditional methods whichmay be employed are co-immunoprecipitation, crosslinking andco-purification through gradients or chromatographic columns of celllysates or proteins obtained from cell lysates and the use of ALARM toidentify proteins in the lysate that interact with the ALARM. For theseassays, the ALARM polypetide can be a full length ALARM, a solubleextracellular form of ALARM or some other suitable ALARM polypeptide.Once isolated, such an interacting protein can be identified and clonedand then used, in conjunction with standard techniques, to identifyproteins with which it interacts. For example, at least a portion of theamino acid sequence of a protein which interacts with the ALARM can beascertained using techniques well known to those of skill in the art,such as via the Edman degradation technique. The amino acid sequenceobtained may be used as a guide for the generation of oligonucleotidemixtures that can be used to screen for gene sequences encoding theinteracting protein. Screening may be accomplished, for example, bystandard hybridization or PCR techniques. Techniques for the generationof oligonucleotide mixtures and the screening are well-known. (Ausubel,supra; and PCR Protocols: A Guide to Methods and Applications, 1990,Innis et al., eds. Academic Press, Inc., New York).

Additionally, methods may be employed which result directly in theidentification of genes which encode proteins which interact with ALARM.These methods include, for example, screening expression libraries, in amanner similar to the well known technique of antibody probing of λgt11libraries, using labeled ALARM polypeptide or an ALARM fusion protein,e.g., an ALARM polypeptide or domain fused to a marker such as anenzyme, fluorescent dye, a luminescent protein, or to an IgFc domain.

The method used to identify the ALARM protein, described below, based onits interaction with presenilin 1 (see also Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578, 1991) can also be used to detect other proteinsinteracting with ALARM. A kit for practicing this method is availablefrom Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid includes a nucleotide sequence encodingthe DNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding an ALARM polypeptide or protein, or anALARM fusion protein, and the other plasmid includes a nucleolidesequence encoding the transcription activator protein's activationdomain fused to a cDNA encoding an unknown protein which has beenrecombined into this plasmid as part of a cDNA library. The DNA-bindingdomain fusion plasmid and the cDNA library are transformed into a strainof the yeast Saccharomyces cerevisiae that contains a reporter gene(e.g., HBS or lacZ) whose regulatory region contains the transcriptionactivator's binding site. Either hybrid protein alone cannot activatetranscription of the reporter gene: the DNA-binding domain hybrid cannotbecause it does not provide activation function and the activationdomain hybrid cannot because it cannot localize to the activator'sbinding sites. Interaction of the two hybrid proteins reconstitutes thefunctional activator protein and results in expression of the reportergene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, ALARM maybe used as the bait gene product. Total genomic or cDNA sequences arefused to the DNA encoding an activation domain. This library and aplasmid encoding a hybrid of bait ALARM gene product fused to theDNA-binding domain are cotransformed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene. For example, a bait ALARM gene sequence, such as ALARM ora domain of ALARM can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait ALARM gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait ALARM gene-GAL4 fusion plasmid into a yeast strain whichcontains a lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait ALARM gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies which express HIS3 can then be purified from thesestrains, and used to produce and isolate the bait ALARM gene-interactingprotein using techniques routinely practiced in the art.

In addition, a genetic test can also be used wherein ALARM nucleic acidsequences are used to identify polymorphisms in the ALARM gene whichindicate an increased likelihood of developing a condition or disease.

Diagnosis of Diseases Associated with Alterations in ALARM Nucleic AcidSequences

The invention disclosed herein also relates to diagnosis of variousdiseases by first identifying the genetic defect in ALARM which causesthe disease in question, and then devising an assay using either ahybridization probe or a PCR amplification primer containing the mutantsequence.

After identifying a specific ALARM mutation that is associated with aparticular disease, that information can then be used to design anoligonucleotide useful as a diagnostic tool to screen other individualsfor that particular disease.

The oligonucleotide can take the form of a hybridization probe or aprimer for PCR amplification. Such hybridization probes could range insize from six to 10,000 nucleotides (preferably 13 to 20 nucleotides),while PCR primers could range from ten to 1000 nucleotides (preferably18 to 25 nucleotides).

If either such screen reveals that the mutation appears in some patientswith an autosomal dominant disease but in no unaffected individuals of astatistically significant sample, it can be presumed that the existenceof that mutation in the DNA of any tested individual will be informativefor the inherited propensity to develop one form of autosomal dominantALARM-protein related disease. An oligonucleotide which includes themutant sequence will be useful as a diagnostic tool for screeningindividuals for that form of the disease. A genetic screening test basedon this oligonucleotide, and further including a second oligonucleotidewith the normal sequence could be useful not only to detect thosehomozygous for the mutation (and thus destined to develop the disease),but also those heterozygous for the mutation (and thus carriers of thedisease trait).

A genetic screening test can also be used to identify individuals withautosomal recessive ALARM-associated disease, and/or to identifycompound heterozygotes. In the latter case, two different mutations,each affecting different copies of the disease gene, are present in theaffected patients of a sibship. Each of the two mutations comes from oneparent.

Uses of the Invention

The ALARM proteins and nucleic acids of the invention have a variety ofuses. For example, an ALARM polypeptide can be used to determine theamount of ALARM-binding presenilin 1 in a sample.

In addition, ALARM antibodies can be used in an immunoassay to monitorthe level of ALARM produced by a mammal and also to determine thesubcellular location of ALARM in a mammal.

Further, both ALARM polypeptides and ALARM antibodies can be used toidentify additional proteins which bind to ALARM.

ALARM nucleic acids can also be used to identify human chromosome 5, asdiagnostic agents to identify individuals with mutations in ALARMnucleic acid sequences. In addition, ALARM nucleic acid and polypeptidesequences be used as molecular weight markers and also to blockexpression of ALARM sequences.

EXAMPLES

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims. Thefollowing examples illustrate the characterization of human ALARMnucleic acids and polypeptides.

Example 1 Primary Structure of ALARM Nucleic Acids and Polypeptides

The yeast two-hybrid system was used to identify cDNAs from a humanbrain cDNA library that bind to the Loop region of presenilin 1.

PCR was used to amplify the presenilin 1 loop region, which is definedby EcoRI and BamHI sites at it 5′ or 3′ ends, respectively and whichencodes amino acids 260-400 of presenilin. The PCR products and vectorPAS2-1 DNA were digested with these two restriction enzymes and thenligated. The resulting construct was confirmed by sequence analysis andnamed the Loop construct.

Standard procedures were used to identify brain. cDNAS that encodedproteins binding to the Loop region. Briefly, the plasmid DNA for thecontrols of the yeast two hybrid experiment was from the MATCHMAKER IIkit (Clontech) which includes pCL1 (full length Gal 4), pVA3-1 (P53 toGal 4 binding domain), pTD1-l (SV40 large T-antigen to activationdomain), pLAM 5′-1 (Human lamin C to BD).

Plasmid DNAs were introduced into yeast strain 190, Strain 190 wastransformed first with the Loop plasmid and with a human brain cDNAlibrary (the Matchmaker library). In each case, selection fortransformants was made on appropriate selective medium.

Plasmid DNA from presumptive colonies containing an interacting cDNA wasisolated from a single, well-isolated colony using standard procedures.The plasmid DNA was then transformed into E. coli, from which plasmidDNA was prepared.

Using the Loop region as bait, eight colonies were identified in the twohybrid assay. Two colonies were positive upon rescreening, and they werefound to carry the identical insert.

To verify that the interaction between the “captured protein” and theLoop region was not an artifact of the two hybrid system, the insert wastranscribed and translated in vitro, and the resulting protein wastested for its ability to bind a Loop-glutathione S-transferase protein.

Fresh overnight cultures of E. coli transformed with pGex-4T-1 or one ofits recombinants were diluted 1:10 in LB-Amp and incubated for 2 hoursat 37C. with shaking until the A_(6C0) reached 0.6-1.0. IPTG was addedto a final concentration of 0.1 mM and the culture was incubated for anadditional 3hours. The cells were washed once with PBS, and resuspendedin 1 ml PBS plus protease inhibitors (PMSF, aprotinin, leupeptin,pepstatin) in microfuge tubes and then lysed by mild sonication. TritonX-100 in PBS was then added to a final concentration of 1%. The lysatewas rotated at 4° C. for 20 minutes followed by centrifugation at 14,000for 10 minutes at 4° C.

The supernatant was rocked for 15-30 minutes at 4° C. with 20 μl of 50%(v/v) glutathione-sepharose, which had been previously washed with PBS.After centrifugation, the beads were washed three times with PBS.

In vitro translation was performed using Promega (Madison, Wis.) TNTkits. Briefly, 1-2 ug of plasmid DNA was mixed with 25 ul of TnT rabbitreticulocyte lysate, 2 ul reaction buffer, 1 ul T7 RNA polymerase, 2 ulamino acid mixture minus methionine, 4 ul ³⁵S-methionine, 1 ul Rnasinand H₂O in a 50 ul reaction volume. The reaction was incubated at 30° C.for 2 hours.

In vitro translated proteins were mixed in binding buffer (10 mMTris-HCl, pH 8.0; 200 mM NaCl; 5 mM EDTA, 0.5% NP-40, 1 mM DTT, 3 mg/mlof BSA, and proteinase inhibitors) with 20 ul of protein A agarose androcked at 4° C. for 1 hour. Antibody was added to the preclearedsupernatant (1:200), and rocked for 2 hours at 40° C. and then 20 ul ofprotein-A agarose was added and rocked for another 2 hours. The beadswere washed then washed 4 times.

Glutathione-sepharose beads bound with GST-fusion proteins were washedwith binding buffer (10 mM Tris-HC1, pH 8.0; 200 mM NaCl; 5 mM EDTA,0.5% NP-40, 1 mM DTT, 3 mg/ml of BSA, and proteinase inhibitors), rockedwith aliquots of in vitro translated ³⁵S-labeled proteins for 1 hour at4° C. in binding buffer. The beads were washed five times with bindingbuffer and boiled in sample buffer. The eluted proteins were thenanalyzed on SDS-PAGE. Binding of the captured protein to theLoop-glutathione S-transferase protein was observed, confirming that thecaptured protein was not an artifact of the yeast two-hybrid system.

The DNA encoding the captured protein was then sequenced. Sequenceanalysis was performed using the GCG sequence analysis program.

The captured protein was found to have the DNA sequence shown in FIG. 1SEQ ID NO:1, and encode a protein with the amino acid sequence shown inFIG. 1 SEQ ID NO:2. The protein contains four copies of a the armrepeat, which was originally identified in the Drosophila melanogasterarm gene and has been subsequently identified in members of the cateninfamily. Because members of the catenin family have been associated withthe adherens junctions, the new protein has been named ALARM, foradherens-junction linked arm protein, or, alternatively, δ catenin. Thepresence of the arm repeats in the ALARM protein, and their similarityto the original arm repeat is shown in FIG. 2. The arm repeats from theALARM sequence are represented labeled as 1 SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, and SEQ ID NO:7. Repeat ii SEQ ID NO:5 is most homologousto arm, SEQ ID NO:3 with 70% homology, while repeat iii SEQ ID NO:6 isthe least homologous, with 31% homology.

Among proteins identified which contain arm repeats, ALARM shows thehighest homology to ppl20, a protein originally identified as asubstrate for the tyrosine kinase pp6osrc (Staddon et al., J. Cell Biol.130:369, 1995). The. ppl120 homology is shown in FIG. 3. Overall, ALARMSEQ ID NO:8 is 60.8% similar and 43.3% identical to ppl120SEQ ID NO:9.

The sequence alignment between ALARM and γ catenin, is shown in FIG. 4.Overall, ALARM SEQ ID NO:10 and γ catenin SEQ ID NO:11 are 52.3% similarand 32.1% identical.

In chromosomal mapping studies, DNA sequences homologous toALARM-encoding DNA sequences were found to map to chromosome 5.

Example 2 Tissue Localization of ALARM RNA Sequences

To determine the tissues in which ALARM sequences are transcribed, polyA⁺ RNA was isolated from several human tissues.

RNA was isolated from human tissues using standard procedures. RNAhybridization was performed using Clontech ExpressHyb solution. Briefly,the ExpressHyb Solution was warmed up to 60° C. The nylon membrane wasprehybridized in 5 ml of ExpressHyb Solution with continuous shaking at60° C. for 30 minutes. Denatured ALARM DNA (labeled with ³²P by randomprimer extension) was added to 5 ml of fresh ExpressHyb to a finalactivity 10⁶ cpm/ml, and the hybridization was carried out for 1 hour at60° C. The blot was rinsed in wash solution 1 several times at roomtemperature for 30-40 minutes with continuous agitation, and then washedin wash solution 2 with continuous shaking at 500C for 40 minutes withone change of fresh solution. The blot was then exposed to x-ray film at−70° C. with two intensifying screens.

An intensely hybridizing band 6 kb in size was detected in tissue frombrain, as were minor bands of 7 kb and 4.0kb. A weak to moderatelyhybridizing band of 6 kb was detected in the pancreas. Heart tissue gaverise to barely detectable transcripts, and no hybridization was detectedin skeletal muscle, kidney, liver, placenta, or lung. These dataindicate that ALARM expression is found nearly exclusively in braintissue.

Example 3 Generation of Antibodies Against ALARM Peptides andCo-immunoprecipitation Experiments Using ALARM Anti-Sera

Polyclonal anti-ALARM antibodies were raised using standard proceduresby injecting a synthetic 14 amino acid peptide having the sequenceYETSHYPASPDSWVSEQ ID NO:13, corresponding to the 14 carboxy terminalresidues of the ALARM protein,into rabbits. Anti-alarm antibodies werealso raised against a GST-fusion protein containing the 100 aminoterminal amino acids of ALARM as shown in FIG. 1 SEQ ID NO:2. Antibodiesraised to the peptides detected a protein migrating with a size of about130 kDa.

To determine if ALARM binds cadherin or the β-amyloid precursor protein(βAPP) protein, co-immunoprecipitation experiments were performed inwhich anti-ALARM sera was used in co-immunoprecipitation experimentsusing ALARM and each of the respective proteins. Anti-ALARM seraprecipitated cadherin protein when ALARM and cadherin proteins werecoexpressed in vitro. Anti-Alarm anti-sera also immunoprecipitatedcadherin in isolated brain tissues. This suggests that ALARM andcadherin interact directly. In addition, because cadherin is found atthe adherens junction, it indicates ALARM also localizes to thisstructure.

Anti ALARM sera also co-precipitated the βAPP precursor peptide whenthese proteins were coexpressed in vitro. This result suggests thatALARM and the APPβ protein bind directly, and that ALARM may be involvedin generating the Aβ peptide.

Example 4 Cellular Localization of ALARM Polypeptides

Immunolocalization studies examining ALARM expression in neuronscultured from embryonic day 18 rat brains were performed using ananti-ALARM antibody isolated as described in Example 3. Rat brainsshowed neuronal staining primarily in the cell body. The observedpattern is consistent with the reported expression pattern of presenilin1.

Example 5 Diagnostic Assays Utilizing ALARM Hybridization Probes

As described above, a nucleic acid probe containing some or all of theALARM-encoding sequences of the invention is used to detect ALARM mRNAin a sample of cells (e.g., brain cells) suspected of having alteredALARM expression. The probe used is a single-stranded DNA or RNA(preferably DNA) antisense to the ALARM coding sequence. It is producedby synthetic or recombinant DNA methods, and labelled with a radioactivetracer or other standard detecting means. The probe includes from 15 upto the full ALARM coding sequence, and preferably is at least 30nucleotides long. The assay is carried out by standard methods of insitu hybridization or Northern analysis, using stringent hybridizationconditions. Control hybridization assays are run in parallel usingnormal cells or tissue sections from the same type of tissue as the testsample, and/or cells from a known tissue or cell line, or a tissuesection, whose ALARM transcription levels are known. Cells which exhibitan altered level of hybridization to the probe, compared to the levelseen with normal epithelial cells, are likely to be indicative of aneurological condition. The amount of hybridization is quantitated bystandard methods, such as counting the grains of radioactivity-exposedemulsion on an in situ hybridization assay of a biopsy slide, or bydensitometric scan of a Northern blot X-ray film. Alternatively,comparison of the test assay results with the results of the controlassays is relative rather than quantitative, particularly where thedifference in levels of hybridization is dramatic.

Example 6 Diagnostic Assays Utilizing Alarm Antibodies

Antibodies specific for ALARM are generated by standard polyclonal ormonoclonal methods, using as immunogen a purified, naturally-occurringALARM; recombinant ALARM; or any antigenic fragment of ALARM (e.g., thepeptides described above) which induces antibodies that react withnaturally-occurring ALARM. The latter fragment can be produced bysynthetic or recombinant methods, or by proteolytic digestion of ALARM.If desired, the antigenic fragment is linked by standard methods to amolecule which increases the immunogenicity of the fragment, such askeyhole limpet hemocyanin (as described above). The polyclonal ormonoclonal antibodies so produced are screened using purifiedrecombinant or naturally occurring ALARM, or as described above, toselect those which form an immunocomplex with ALARM specifically.

The antibodies so produced are employed in diagnostic methods fordetecting cells, tissues, or biological fluids in which the presence ofALARM is altered relative to normal cells, an indication that thepatient has a neurological condition. The sample tested may be a fixedsection of a tissue biopsy, a preparation of cells obtained from asuspect tissue, or a sample of biological fluid, such as cerebrospinalfluid. Standard methods of immunoassay may be used, including thosedescribed above as well as sandwich ELISA. If the tested cells expressaltered levels of ALARM protein in this assay relative to normal cellsof the same tissue type, the tested cells are likely to represent aneurological condition. The anti-ALARM antibodies are also used todetect alterations in the levels or ALARM-binding activity of othercellular components, e.g., presenilin-1, cadherein, or βAPP protein,that interact with ALARM. Anti-ALARM anti-bodies are used to detectthese proteins using co-immunoprecipitation assays known in the art.

Example 7 Screens for and Uses of Therapeutic Agents Based on theirInteraction with ALARM

Cells in which the expression or activity of the endogenous ALARM geneis altered, i.e., down-regulated, are used as a screening tool toidentify compounds or treatment strategies that increase expression oractivity of the ALARM gene.

The cells are treated in vitro with the candidate compounds, and theamount of ALARM expression is determined using either a hybridizationassay (e.g., Northern analysis) or an immunoassay. If a given compoundis found to increase ALARM expression, it is then further tested to seewhether treatment with the compound prevents the development of aneurological condition in vivo in an appropriate animal model. Anappropriate animal model is a transgenic model constructed using thetechniques described above in which a ALARM gene is expressed under thecontrol of an inducible promoter.

A compound effective both in increasing ALARM expression or activity(e.g., in facilitating its binding to presenilin 1 or βAPP) is apotential therapeutic useful for the treatment of conditions in whichALARM expression is increased compared to normal cells. Furtherevaluation of the clinical usefulness of such a compound followsstandard methods of evaluating toxicity and clinical effectiveness ofagents for treating neurological conditions.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

13 2983 base pairs nucleic acid single linear cDNA Coding Sequence366...2633 1 GCACCAGCTC GCCCATCAAC ATCGTCGTGT CCTCGGCCGG CCTGTCCCCGATCCGCGTGA 60 CCTCGCCCCC CACCGTGCAG TCCACCATCT CCTCCTCGCC CATCCACCAGCTGAGCTCCA 120 CCATCGGCAC GTACGCCACC CTGTCGCCCA CCAAGCGCCT GGTCCACGCGTCCGAGCAGT 180 ACAGCAAGCA CTCGCAGGAG CTGTATGCCA CGGCCACCCT CCAGAGGCCGGGCAGCCTGG 240 CAGCTGGTTC CCGAGCCTCA TACAGCAGCC AGCATGGGCA CCTGGGCCCAGAGTTGCGGG 300 CCCTGCAGTC CCCAGAACAC CACATAGATC CCATCTATGA AGTCCGCGTCTATCAGAAGC 360 CCCCT ATG AGG AGT CTC AGC CAG AGC CAG GGG GTC CCT CTG CCGCCA GCA 410 Met Arg Ser Leu Ser Gln Ser Gln Gly Val Pro Leu Pro Pro Ala1 5 10 15 CAC ACC GGC ACC TAC CGC ACG AGC ACA GCC CCA TCT TCC CCT GGTGTC 458 His Thr Gly Thr Tyr Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val20 25 30 GAC TCC GTC CCC TTG CAG CGC ACA GGC AGC CAG CAC GGC CCA CAG AAT506 Asp Ser Val Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn 3540 45 GCC GCC GCG GCC ACC TTC CAG AGG GCC AGC TAT GCC GCC GGC CCA GCC554 Ala Ala Ala Ala Thr Phe Gln Arg Ala Ser Tyr Ala Ala Gly Pro Ala 5055 60 TCC AAT TAC GCG GAC CCC TAC CGA CAG CTG CAG TAT TGT CCC TCT GTT602 Ser Asn Tyr Ala Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Pro Ser Val 6570 75 GAG TCT CCA TAC AGC AAA TCC GGC CCT GCT CTC CCG CCT GAA GGC ACC650 Glu Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr 8085 90 95 TTG GCC AGG TCC CCG TCC ATT GAT AGC ATT CAG AAA GAT CCC AGA GAA698 Leu Ala Arg Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu 100105 110 TTT GGA TGG AGA GAC CCG GAA CTG CCG GAA GTG ATT CAG ATG TTG CAG746 Phe Gly Trp Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln 115120 125 CAC CAG TTT CCC TCG GTC CAG TCT AAC GCG GCA GCC TAC TTG CAA CAC794 His Gln Phe Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His 130135 140 CTC TGT TTT GGA GAC AAC AAA ATT AAA GCC GAG ATA AGG AGA CAA GGA842 Leu Cys Phe Gly Asp Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly 145150 155 GGC ATC CAG CTC CTG GTG GAC CTG TTG GAT CAT CGG ATG ACC GAA GTC890 Gly Ile Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu Val 160165 170 175 CAC CGT AGT GCC TGT GGA GCT CTG AGA AAC CTG GTG TAT GGG AAGGCC 938 His Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala180 185 190 AAC GAT GAT AAC AAA ATT GCC CTG AAA AAC TGT GGT GGC ATC CCAGCA 986 Asn Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro Ala195 200 205 CTG GTG AGG TTA CTC CGC AAG ACG ACT GAC CTG GAG ATC CGG GAGCTG 1034 Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu210 215 220 GTC ACA GGA GTC CTT TGG AAC CTC TCC TCA TGC GAT GCA CTC AAAATG 1082 Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met225 230 235 CCA ATC ATC CAG GAT GCC CTA GCA GTA CTG ACC AAC GCG GTG ATTATC 1130 Pro Ile Ile Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile240 245 250 255 CCC CAC TCA GGC TGG GAA AAT TCG CCT CTT CAG GAT GAT CGGAAA ATA 1178 Pro His Ser Gly Trp Glu Asn Ser Pro Leu Gln Asp Asp Arg LysIle 260 265 270 CAG CTG CAT TCA TCA CAG GTG CTG CGT AAC GCC ACC GGG TGCTTA AGG 1226 Gln Leu His Ser Ser Gln Val Leu Arg Asn Ala Thr Gly Cys LeuArg 275 280 285 AAT GTT AGT TCG CCC GGA GAG GAG GCC CGC AGA AGG ATG AGAGAG TGT 1274 Asn Val Ser Ser Pro Gly Glu Glu Ala Arg Arg Arg Met Arg GluCys 290 295 300 GAT GGG CTT ACG GAT GCC TTG CTG TAC GTG ATC CAG TCT GCGCTG GGG 1322 Asp Gly Leu Thr Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala LeuGly 305 310 315 AGC AGT GAG ATC GAT AGC AAG ACC GTT GAA AAC TGT GTG TGCATT TTA 1370 Ser Ser Glu Ile Asp Ser Lys Thr Val Glu Asn Cys Val Cys IleLeu 320 325 330 335 AGG AAC CTC TCG TAC CGG CTG GCG GCA GAA ACG TCT CAGGGA CAG CAC 1418 Arg Asn Leu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln GlyGln His 340 345 350 ATG GGC ACG GAC GAG CTG GAC GGG CTA CTC TGT GGC GAGGCC AAT GGC 1466 Met Gly Thr Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu AlaAsn Gly 355 360 365 AAG GAT GCT GAG AGC TCT GGG TGC TGG GGC AAG AAG AAGAAG AAA AAG 1514 Lys Asp Ala Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys LysLys Lys 370 375 380 AAA TCC CAA GAT CAG TGG GAT GGA GTA GGA CCT CTT CCAGAC TGT GCT 1562 Lys Ser Gln Asp Gln Trp Asp Gly Val Gly Pro Leu Pro AspCys Ala 385 390 395 GAA CCA CCA AAA GGG ATC CAG ATG CTG TGG CAC CCA TCAATA GTC AAA 1610 Glu Pro Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser IleVal Lys 400 405 410 415 CCC TAC CTC ACA CTG CTC TCT GAG TGC TCA AAT CCAGAC ACG CTG GAA 1658 Pro Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro AspThr Leu Glu 420 425 430 GGG GCG GCA GGC GCC CTG CAG AAC TTG GCT GCA GGGAGC TGG AAG TGG 1706 Gly Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly SerTrp Lys Trp 435 440 445 TCA GTA TAT ATC CGA GCC GCT GTC CGA AAA GAG AAAGGC CGG CCC ATC 1754 Ser Val Tyr Ile Arg Ala Ala Val Arg Lys Glu Lys GlyArg Pro Ile 450 455 460 CTC GTG GAG CTG CTC CGA ATA GAC AAT GAC CGT GTGGCG TGC GCG GTG 1802 Leu Val Glu Leu Leu Arg Ile Asp Asn Asp Arg Val AlaCys Ala Val 465 470 475 GCC ACT GCG CTG CGG AAC ATG GCC TTG GAC GTC AGAAAT AAG GAG CTC 1850 Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg AsnLys Glu Leu 480 485 490 495 ATC GGC AAA TAC GCC ATG CGA GAC CTA GTC CACAGG CTT CCA GGA GGG 1898 Ile Gly Lys Tyr Ala Met Arg Asp Leu Val His ArgLeu Pro Gly Gly 500 505 510 AAC AAC AGC AAC AAC ACT GCA AGC AAG GCC ATGTCG GAT GAC ACA GTG 1946 Asn Asn Ser Asn Asn Thr Ala Ser Lys Ala Met SerAsp Asp Thr Val 515 520 525 ACA GCT GTC TGC TGC ACA CTG CAC GAA GTG ATTACC AAG AAC ATG GAG 1994 Thr Ala Val Cys Cys Thr Leu His Glu Val Ile ThrLys Asn Met Glu 530 535 540 AAC GCC AAG GCC TTA CGG GAT GCC GGT GGC ATCGAG AAG TTG GTC GGC 2042 Asn Ala Lys Ala Leu Arg Asp Ala Gly Gly Ile GluLys Leu Val Gly 545 550 555 ATC TCC AAA AGC AAA GGA GAT AAA CAC TCT CCAAAA GTG GTC AAG GCT 2090 Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro LysVal Val Lys Ala 560 565 570 575 GCA TCT CAG GTC CTC AAC AGC ATG TGG CAGTAC CGA GAT CTG AGG AGT 2138 Ala Ser Gln Val Leu Asn Ser Met Trp Gln TyrArg Asp Leu Arg Ser 580 585 590 CTC TAC AAA AAG GAT GGA TGG TCA CAA TACCAC TTT GTA GCC TCG TCT 2186 Leu Tyr Lys Lys Asp Gly Trp Ser Gln Tyr HisPhe Val Ala Ser Ser 595 600 605 TCA ACC ATC GAG AGG GAC CGG CAA AGG CCCTAC TCC TCC TCC CGC ACG 2234 Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro TyrSer Ser Ser Arg Thr 610 615 620 CCC TCC ATC TCC CCT GTG CGC GTG TCT CCCAAC AAC CGC TCA GCA AGT 2282 Pro Ser Ile Ser Pro Val Arg Val Ser Pro AsnAsn Arg Ser Ala Ser 625 630 635 GCC CCA GCT TCA CCT CGG GAA ATG ATC AGCCTC AAA GAA AGG AAA ACA 2330 Ala Pro Ala Ser Pro Arg Glu Met Ile Ser LeuLys Glu Arg Lys Thr 640 645 650 655 GAC TAC GAG TGC ACC GGC AGC AAC GCCACC TAC CAC GGA GCT AAA GGC 2378 Asp Tyr Glu Cys Thr Gly Ser Asn Ala ThrTyr His Gly Ala Lys Gly 660 665 670 GAA CAC ACT TCC AGG AAA GAT GCC ATGACA GCT CAA AAC ACT GGA ATT 2426 Glu His Thr Ser Arg Lys Asp Ala Met ThrAla Gln Asn Thr Gly Ile 675 680 685 TCA ACT TTG TAT AGG AAT TCT ACA AGAAAT TAC GAT GAG TCC TTC TTC 2474 Ser Thr Leu Tyr Arg Asn Ser Thr Arg AsnTyr Asp Glu Ser Phe Phe 690 695 700 GAG GAC CAG GTC CAC CAT CGC CCT CCCGCC AGC GAG TAC ACC ATG CAC 2522 Glu Asp Gln Val His His Arg Pro Pro AlaSer Glu Tyr Thr Met His 705 710 715 CTG GGT CTC AAG TCC ACC GGC AAC TACGTT GAC TTC TAC TCA GCT GCC 2570 Leu Gly Leu Lys Ser Thr Gly Asn Tyr ValAsp Phe Tyr Ser Ala Ala 720 725 730 735 CGT CCC TAC AGT GAA CTG AAC TATGAA ACG AGC CAC TAC CCG GCC TCC 2618 Arg Pro Tyr Ser Glu Leu Asn Tyr GluThr Ser His Tyr Pro Ala Ser 740 745 750 CCC GAC TCC TGG GTG TGAGGAGCAGGGCACAGGCG CTCCGGGAAC AGTGCATGTG 2673 Pro Asp Ser Trp Val 755 CATGCATACCACAAGACATT TCTTTCTGTT TTGTTTTTTT CTCCTGCAAA TTTAGTTTGT 2733 TAAAGCCTGTTCCATAGGAA GGCTGTGATA ACCAGTAAGG AAATATTAAG AGCTATTTTA 2793 GAAAGCTAAATGAATCGCAA GTTTAACTTG GAAATCAGTA GAAAGCTAAA GTGATCCTAA 2853 ATATGACAGTGGGCAGCACC TTTCTAGCGT GAGCTGTAAA GTAACGANAA GTGCTTTATA 2913 CTGAACGTNGTTGATGGGAG GANANACAAG CATTCCGGCC GGTGGGGCNT ANGGTTNTCN 2973 TTAACACAAT2983 756 amino acids amino acid linear protein internal 2 Met Arg SerLeu Ser Gln Ser Gln Gly Val Pro Leu Pro Pro Ala His 1 5 10 15 Thr GlyThr Tyr Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp 20 25 30 Ser ValPro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gln Asn Ala 35 40 45 Ala AlaAla Thr Phe Gln Arg Ala Ser Tyr Ala Ala Gly Pro Ala Ser 50 55 60 Asn TyrAla Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Pro Ser Val Glu 65 70 75 80 SerPro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu 85 90 95 AlaArg Ser Pro Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu Phe 100 105 110Gly Trp Arg Asp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln His 115 120125 Gln Phe Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Leu 130135 140 Cys Phe Gly Asp Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly Gly145 150 155 160 Ile Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr GluVal His 165 170 175 Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr GlyLys Ala Asn 180 185 190 Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly GlyIle Pro Ala Leu 195 200 205 Val Arg Leu Leu Arg Lys Thr Thr Asp Leu GluIle Arg Glu Leu Val 210 215 220 Thr Gly Val Leu Trp Asn Leu Ser Ser CysAsp Ala Leu Lys Met Pro 225 230 235 240 Ile Ile Gln Asp Ala Leu Ala ValLeu Thr Asn Ala Val Ile Ile Pro 245 250 255 His Ser Gly Trp Glu Asn SerPro Leu Gln Asp Asp Arg Lys Ile Gln 260 265 270 Leu His Ser Ser Gln ValLeu Arg Asn Ala Thr Gly Cys Leu Arg Asn 275 280 285 Val Ser Ser Pro GlyGlu Glu Ala Arg Arg Arg Met Arg Glu Cys Asp 290 295 300 Gly Leu Thr AspAla Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly Ser 305 310 315 320 Ser GluIle Asp Ser Lys Thr Val Glu Asn Cys Val Cys Ile Leu Arg 325 330 335 AsnLeu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met 340 345 350Gly Thr Asp Glu Leu Asp Gly Leu Leu Cys Gly Glu Ala Asn Gly Lys 355 360365 Asp Ala Glu Ser Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys Lys 370375 380 Ser Gln Asp Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu385 390 395 400 Pro Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile ValLys Pro 405 410 415 Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp ThrLeu Glu Gly 420 425 430 Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly SerTrp Lys Trp Ser 435 440 445 Val Tyr Ile Arg Ala Ala Val Arg Lys Glu LysGly Arg Pro Ile Leu 450 455 460 Val Glu Leu Leu Arg Ile Asp Asn Asp ArgVal Ala Cys Ala Val Ala 465 470 475 480 Thr Ala Leu Arg Asn Met Ala LeuAsp Val Arg Asn Lys Glu Leu Ile 485 490 495 Gly Lys Tyr Ala Met Arg AspLeu Val His Arg Leu Pro Gly Gly Asn 500 505 510 Asn Ser Asn Asn Thr AlaSer Lys Ala Met Ser Asp Asp Thr Val Thr 515 520 525 Ala Val Cys Cys ThrLeu His Glu Val Ile Thr Lys Asn Met Glu Asn 530 535 540 Ala Lys Ala LeuArg Asp Ala Gly Gly Ile Glu Lys Leu Val Gly Ile 545 550 555 560 Ser LysSer Lys Gly Asp Lys His Ser Pro Lys Val Val Lys Ala Ala 565 570 575 SerGln Val Leu Asn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser Leu 580 585 590Tyr Lys Lys Asp Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser 595 600605 Thr Ile Glu Arg Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro 610615 620 Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala625 630 635 640 Pro Ala Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg LysThr Asp 645 650 655 Tyr Glu Cys Thr Gly Ser Asn Ala Thr Tyr His Gly AlaLys Gly Glu 660 665 670 His Thr Ser Arg Lys Asp Ala Met Thr Ala Gln AsnThr Gly Ile Ser 675 680 685 Thr Leu Tyr Arg Asn Ser Thr Arg Asn Tyr AspGlu Ser Phe Phe Glu 690 695 700 Asp Gln Val His His Arg Pro Pro Ala SerGlu Tyr Thr Met His Leu 705 710 715 720 Gly Leu Lys Ser Thr Gly Asn TyrVal Asp Phe Tyr Ser Ala Ala Arg 725 730 735 Pro Tyr Ser Glu Leu Asn TyrGlu Thr Ser His Tyr Pro Ala Ser Pro 740 745 750 Asp Ser Trp Val 755 44amino acids amino acid linear peptide 11, 13-14, 16-18, 21, 23, 33-36,and 41-44 where Xaa at positions 11, 13-14, 16-18, 21, 23, 33-36, and41-44 is any amino acid 3 Gly Gly Ile Pro Ala Leu Val Arg Leu Leu XaaAsn Xaa Xaa Asp Xaa 1 5 10 15 Xaa Xaa Leu Leu Xaa Ala Ala Xaa Gly ValLeu Arg Asn Leu Ser Xaa 20 25 30 Xaa Xaa Xaa Xaa Asn Lys Ala Ile Xaa XaaXaa Xaa 35 40 44 amino acids amino acid linear peptide 4 Gly Gly Ile GlnLeu Leu Val Asp Leu Leu Asp His Arg Met Thr Glu 1 5 10 15 Val His ArgSer Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys 20 25 30 Ala Asn AspAsp Asn Lys Ile Ala Leu Lys Asn Cys 35 40 41 amino acids amino acidlinear peptide 5 Gly Gly Ile Pro Ala Leu Val Arg Leu Leu Arg Lys Thr ThrAsp Leu 1 5 10 15 Glu Ile Arg Glu Leu Val Thr Gly Val Leu Trp Asn LeuSer Ser Cys 20 25 30 Asp Ala Leu Lys Met Pro Ile Ile Gln 35 40 39 aminoacids amino acid linear peptide 6 Ser Ile Val Lys Pro Tyr Leu Thr LeuLeu Ser Glu Cys Ser Asn Pro 1 5 10 15 Asp Thr Leu Glu Gly Ala Ala GlyAla Leu Gln Asn Leu Ala Ala Gly 20 25 30 Ser Trp Lys Trp Ser Val Tyr 3541 amino acids amino acid linear peptide 7 Lys Gly Arg Pro Ile Leu ValGlu Leu Leu Arg Ile Asp Asn Asp Arg 1 5 10 15 Val Ala Cys Ala Val AlaThr Ala Leu Arg Asn Met Ala Leu Asp Val 20 25 30 Arg Asn Lys Glu Leu IleGly Lys Tyr 35 40 686 amino acids amino acid linear protein 8 Ser GlnSer Gln Gly Val Pro Leu Pro Pro Ala His Thr Gly Thr Tyr 1 5 10 15 ArgThr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser Val Pro Leu 20 25 30 GlnArg Thr Gly Ser Gln His Gly Pro Gln Asn Ala Ala Ala Ala Thr 35 40 45 PheGln Arg Ala Ser Tyr Ala Ala Gly Pro Ala Ser Asn Tyr Ala Asp 50 55 60 ProTyr Arg Gln Leu Gln Tyr Cys Pro Ser Val Glu Ser Pro Tyr Ser 65 70 75 80Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg Ser Pro 85 90 95Ser Ile Asp Ser Ile Gln Lys Asp Pro Arg Glu Phe Gly Trp Arg Asp 100 105110 Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln His Gln Phe Pro Ser 115120 125 Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Leu Cys Phe Gly Asp130 135 140 Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly Gly Ile Gln LeuLeu 145 150 155 160 Val Asp Leu Leu Asp His Arg Met Thr Glu Val His ArgSer Ala Cys 165 170 175 Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala AsnAsp Asp Asn Lys 180 185 190 Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro AlaLeu Val Arg Leu Leu 195 200 205 Arg Lys Thr Thr Asp Leu Glu Ile Arg GluLeu Val Thr Gly Val Leu 210 215 220 Trp Asn Leu Ser Ser Cys Asp Ala LeuLys Met Pro Ile Ile Gln Asp 225 230 235 240 Ala Leu Ala Val Leu Thr AsnAla Val Ile Ile Pro His Ser Gly Trp 245 250 255 Glu Asn Ser Pro Leu GlnAsp Asp Arg Lys Ile Gln Leu His Ser Ser 260 265 270 Gln Val Leu Arg AsnAla Thr Gly Cys Leu Arg Asn Val Ser Ser Pro 275 280 285 Gly Glu Glu AlaArg Arg Arg Met Arg Glu Cys Asp Gly Leu Thr Asp 290 295 300 Ala Leu LeuTyr Val Ile Gln Ser Ala Leu Gly Ser Ser Glu Ile Asp 305 310 315 320 SerLys Thr Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu Ser Tyr 325 330 335Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr Asp Glu 340 345350 Leu Asp Gly Leu Leu Cys Gly Glu Ala Asn Gly Lys Asp Ala Glu Ser 355360 365 Ser Gly Cys Trp Gly Lys Lys Lys Lys Lys Lys Lys Ser Gln Asp Gln370 375 380 Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro LysGly 385 390 395 400 Ile Gln Met Leu Trp His Pro Ser Ile Val Lys Pro TyrLeu Thr Leu 405 410 415 Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu GlyAla Ala Gly Ala 420 425 430 Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys TrpSer Val Tyr Ile Arg 435 440 445 Ala Ala Val Arg Lys Glu Lys Gly Arg ProIle Leu Val Glu Leu Leu 450 455 460 Arg Ile Asp Asn Asp Arg Val Ala CysAla Val Ala Thr Ala Leu Arg 465 470 475 480 Asn Met Ala Leu Asp Val ArgAsn Lys Glu Leu Ile Gly Lys Tyr Ala 485 490 495 Met Arg Asp Leu Val HisArg Leu Pro Gly Gly Asn Asn Ser Asn Asn 500 505 510 Thr Ala Ser Lys AlaMet Ser Asp Asp Thr Val Thr Ala Val Cys Cys 515 520 525 Thr Leu His GluVal Ile Thr Lys Asn Met Glu Asn Ala Lys Ala Leu 530 535 540 Arg Asp AlaGly Gly Ile Glu Lys Leu Val Gly Ile Ser Lys Ser Lys 545 550 555 560 GlyAsp Lys His Ser Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu 565 570 575Asn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser Leu Tyr Lys Lys Asp 580 585590 Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser Thr Ile Glu Arg 595600 605 Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro610 615 620 Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro Ala SerPro 625 630 635 640 Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr Asp TyrGlu Cys Thr 645 650 655 Gly Ser Asn Ala Thr Tyr His Gly Ala Lys Gly GluHis Thr Ser Arg 660 665 670 Lys Asp Ala Met Thr Ala Gln Asn Thr Gly IleSer Thr Leu 675 680 685 682 amino acids amino acid linear protein 9 SerLeu Ser Arg Val Thr Arg Ile Glu Glu Arg Tyr Arg Pro Ser Met 1 5 10 15Gln Val Arg Val Gly Gly Ser Ser Val Asp Leu His Arg Phe His Pro 20 25 30Gln Val Arg Val Gly Gly Ser Ser Val Asp Leu His Arg Phe His Pro 35 40 45Glu Pro Tyr Gly Leu Glu Asp Asp Gln Arg Ser Met Gly Tyr Asp Asp 50 55 60Leu Asp Tyr Gly Met Met Ser Asp Tyr Gly Thr Ala Arg Arg Thr Gly 65 70 7580 Thr Pro Ser Asp Pro Arg Arg Arg Leu Arg Ser Thr Glu Asp Met Ile 85 9095 Gly Glu Glu Val Pro Pro Asp Gln Tyr Tyr Trp Ala Pro Leu Ala Gln 100105 110 His Glu Arg Gly Ser Leu Ala Ser Leu Asp Ser Leu Arg Lys Gly Met115 120 125 Pro Pro Pro Ser Asn Trp Arg Gln Pro Glu Leu Pro Glu Val IleAla 130 135 140 Met Leu Gly Phe Arg Leu Asp Ala Val Lys Ser Asn Ala AlaAla Tyr 145 150 155 160 Leu Gln His Leu Cys Tyr Arg Asn Asp Lys Val LysThr Asp Val Ala 165 170 175 Lys Leu Lys Gly Ile Pro Ile Leu Val Gly LeuLeu Asp His Pro Lys 180 185 190 Lys Glu Val His Leu Gly Ala Cys Gly AlaLeu Lys Asn Ile Ser Phe 195 200 205 Gly Arg Asp Gln Asp Asn Lys Ile AlaIle Lys Asn Cys Asp Gly Val 210 215 220 Pro Ala Leu Val Arg Leu Leu ArgLys Ala Arg Asp Met Asp Leu Thr 225 230 235 240 Glu Val Ile Thr Gly ThrLeu Trp Asn Leu Ser Ser His Asp Ser Ile 245 250 255 Lys Met Glu Ile ValAsp His Ala Leu His Ala Leu Thr Asp Glu Val 260 265 270 Ile Ile Pro HisSer Gly Trp Glu Arg Glu Pro Asn Glu Asp Cys Lys 275 280 285 Pro Arg HisIle Glu Trp Glu Ser Val Leu Thr Asn Thr Ala Gly Cys 290 295 300 Leu ArgAsn Val Ser Ser Glu Arg Ser Glu Ala Arg Arg Lys Leu Arg 305 310 315 320Glu Cys Asp Gly Leu Val Asp Ala Leu Ile Phe Ile Val Gln Ala Glu 325 330335 Ile Gly Gln Lys Asp Ser Asp Ser Lys Leu Val Glu Asn Cys Val Cys 340345 350 Leu Leu Arg Asn Leu Ser Tyr Gln Val His Arg Glu Ile Pro Gln Ala355 360 365 Glu Arg Tyr Gln Glu Ala Leu Pro Thr Val Ala Asn Ser Thr GlyPro 370 375 380 His Ala Ala Ser Cys Phe Gly Ala Lys Lys Gly Lys Gly LysLys Pro 385 390 395 400 Thr Glu Asp Pro Ala Asn Asp Thr Val Asp Phe ProLys Arg Thr Ser 405 410 415 Pro Ala Arg Gly Tyr Glu Leu Leu Phe Gln ProGlu Val Val Arg Ile 420 425 430 Tyr Ile Ser Leu Leu Lys Glu Ser Asn ThrPro Ala Ile Leu Glu Ala 435 440 445 Ser Ala Gly Ala Ile Gln Asn Leu CysAla Gly Arg Trp Thr Tyr Gly 450 455 460 Arg Tyr Ile Arg Ser Ala Leu ArgGln Glu Lys Ala Leu Ser Ala Arg 465 470 475 480 Ala Glu Leu Leu Thr SerGln His Glu Arg Val Val Lys Ala Ala Ser 485 490 495 Gly Ala Leu Arg AsnLeu Ala Val Asp Ala Arg Asn Lys Glu Leu Ile 500 505 510 Gly Lys His AlaArg Pro Asn Leu Val Lys Asn Leu Pro Gly Gly Gln 515 520 525 Gln Asn SerSer Trp Asn Phe Ser Glu Asp Thr Val Val Ser Ile Leu 530 535 540 Asn ThrIle Asn Glu Val Ile Ala Glu Asn Leu Glu Ala Ala Lys Lys 545 550 555 560Leu Arg Glu Thr Gln Gly Ile Glu Lys Leu Val Leu Ile Asn Lys Ser 565 570575 Gly Asn Arg Ser Glu Lys Glu Val Arg Ala Ala Ala Leu Val Leu Gln 580585 590 Thr Ile Trp Gly Tyr Lys Glu Leu Arg Lys Pro Leu Glu Lys Glu Gly595 600 605 Trp Lys Lys Ser Asp Phe Gln Val Asn Leu Asn Asn Ala Ser ArgSer 610 615 620 Gln Ser Ser His Ser Tyr Asp Asp Ser Thr Leu Pro Leu IleAsp Arg 625 630 635 640 Asn Gln Lys Ser Asp Asn Asn Tyr Ser Thr Leu AsnGlu Arg Gly Asp 645 650 655 His Asn Arg Thr Leu Asp Arg Ser Gly Asp LeuGly Asp Met Glu Pro 660 665 670 Leu Lys Gly Ala Pro Leu Met Gln Lys Ile675 680 620 amino acids amino acid linear protein 10 Arg Ser Leu Ser GlnSer Gln Gly Val Pro Leu Pro Pro Ala His Thr 1 5 10 15 Gly Thr Tyr ArgThr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser 20 25 30 Val Asp Leu GlnArg Thr Cys Ser Gln His Cys Ile Gln Asn Ala Ala 35 40 45 Ala Ala Thr PheGln Arg Ala Cys Tyr Ala Ala Gly Pro Ala Cys Asn 50 55 60 Tyr Ala Asp ProTyr Arg Gln Leu Gln Tyr Cys Pro Ser Val Glu Ser 65 70 75 80 Pro Tyr SerLys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala 85 90 95 Arg Ser ProSer Ile Asp Ser Ile Gln Lys Asp Phe Arg Glu Phe Gly 100 105 110 Trp ArgAsp Pro Glu Leu Pro Glu Val Ile Gln Met Leu Gln Met Gln 115 120 125 PhePro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Leu Cys 130 135 140Phe Gly Asp Asn Lys Ile Lys Ala Glu Ile Arg Arg Gln Gly Gly Ile 145 150155 160 Gln Leu Leu Val Asp Leu Leu Asp His Arg Met Thr Arg Val His Arg165 170 175 Ser Ala Cys Gly Ala Leu Arg Asn Leu Val Tyr Gly Lys Ala AsnAsp 180 185 190 Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro AlaLeu Val 195 200 205 Arg Leu Leu Arg Lys Thr Thr Asp Asp Glu Ile Arg GluLeu Val Thr 210 215 220 Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala LeuLys Met Pro Thr 225 230 235 240 Thr Gln Asp Ala Leu Ala Val Leu Thr AsnAla Val Ile Ile Pro His 245 250 255 Ser Gly Trp Glu Asn Ser Pro Leu GlnAsp Asp Arg Lys Ile Gln Leu 260 265 270 His Ser Ser Gln Val Leu Arg AsnAla Thr Gly Cys Leu Arg Asn Val 275 280 285 Ser Ser Pro Gly Glu Glu AlaArg Arg Arg Met Arg Glu Cys Asp Gly 290 295 300 Leu Thr Asp Ala Leu LeuTyr Val Ile Gln Ser Ala Leu Gly Ser Ser 305 310 315 320 Glu Ile Asp SerLys Thr Val Glu Asn Cys Val Cys Ile Leu Arg Asn 325 330 335 Leu Ser TyrArg Leu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly 340 345 350 Thr AspGlu Leu Asp Gly Leu Leu Cys Cys Glu Ala Asn Cys Phe Asp 355 360 365 AlaGlu Ser Ser Cys Cys Trp Cys Lys Lys Lys Lys Lys Lys Lys Ser 370 375 380Gln Asn Gln Trp Asp Gly Val Gly Pro Leu Pro Asp Cys Ala Glu Pro 385 390395 400 Pro Lys Gly Ile Gln Met Leu Trp His Pro Ser Ile Val Lys Pro Tyr405 410 415 Leu Thr Leu Leu Ser Glu Cys Ser Asn Pro Asp Thr Leu Glu CysAla 420 425 430 Ala Cys Ala Leu Gln Asn Leu Ala Ala Cys Glu Trp Lys TrpGlu Val 435 440 445 Tyr Ile Arg Ala Ala Val Arg Lys Glu Lys Gly Arg ProIle Leu Val 450 455 460 Glu Leu Leu Arg Ile Asp Asn Asp Arg Val Ala CysAla Val Ala Thr 465 470 475 480 Ala Leu Arg Asn Met Ala Leu Asp Val ArgAsn Lys Glu Leu Ile Gly 485 490 495 Lys Tyr Ala Met Arg Asp Leu Val HisArg Leu Pro Gly Gly Asn Asn 500 505 510 Ser Asn Asn Thr Ala Ser Lys AlaMet Ser Asp Asp Thr Val Thr Ala 515 520 525 Val Cys Cys Thr Leu His GluVal Ile Thr Lys Asn Met Glu Asn Ala 530 535 540 Lys Ala Leu Arg Asp AlaGly Gly Ile Glu Lys Leu Val Gly Ile Ser 545 550 555 560 Lys Ser Lys GlyAsp Lys His Ser Pro Lys Val Val Lys Ala Ala Ser 565 570 575 Gln Val LeuAsn Ser Met Trp Gln Tyr Arg Asp Leu Arg Ser Leu Tyr 580 585 590 Lys LysAsp Gly Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser Thr 595 600 605 IleGlu Arg Asp Arg Gln Arg Pro Tyr Arg Arg Arg 610 615 620 666 amino acidsamino acid linear protein 11 Ser Thr Leu Ser Met Ser Asn Arg Gly Ser MetTyr Asp Gly Leu Ala 1 5 10 15 Asp Asn Tyr Asn Tyr Gly Thr Thr Ser LysSer Ser Tyr Tyr Ser Lys 20 25 30 Phe Gln Ala Gly Asn Gly Ser Trp Gly TyrPro Ile Tyr Asn Gly Thr 35 40 45 Leu Lys Arg Glu Pro Asp Asn Arg Arg PheSer Ser Tyr Ser Gln Met 50 55 60 Glu Asn Trp Arg Arg His Tyr Pro Arg GlySer Cys Asn Thr Thr Gly 65 70 75 80 Ala Gly Ser Asp Ile Cys Phe Met GlnLys Ile Lys Ala Ser Arg Ser 85 90 95 Ile Asp Asp Leu Tyr Cys Asp Pro ArgGly Thr Leu Arg Lys Gly Thr 100 105 110 Leu Gly Ser Lys Gly Gln Lys ThrThr Gln Met Arg Tyr Ser Phe Tyr 115 120 125 Ser Thr Cys Ser Gly Gln LysAla Ile Lys Lys Cys Pro Val Arg Pro 130 135 140 Pro Ser Cys Ala Ser LysGln Asp Pro Val Tyr Ile Pro Pro Ile Ser 145 150 155 160 Cys Asn Lys AspLeu Ser Phe Gly Trp Ser Arg Ala Ser Ser Lys Ile 165 170 175 Cys Ser GluAsp Ile Glu Cys Ser Cys Leu Thr Ile Pro Lys Ala Val 180 185 190 Gln TyrLeu Glu Glu Gln Asp Glu Lys Tyr Gln Ala Ile Gly Ala Tyr 195 200 205 TyrIle Gln His Thr Cys Phe Gln Asp Glu Ser Ala Lys Gln Gln Val 210 215 220Tyr Gln Leu Gly Gly Ile Cys Lys Leu Val Asp Leu Leu Arg Ser Pro 225 230235 240 Asn Gln Asn Val Gln Gln Ala Ala Ala Cys Ala Leu Arg Asn Leu Val245 250 255 Phe Arg Glu Thr Thr Asn Lys Leu Glu Thr Arg Arg Gln Asn GlyIle 260 265 270 Arg Glu Ala Val Glu Leu Leu Arg Arg Thr Gly Asn Ala GluIle Gln 275 280 285 Lys Gln Leu Thr Gly Leu Leu Trp Asn Leu Ser Ser ThrAsp Glu Leu 290 295 300 Lys Glu Glu Leu Ile Ala Asp Ala Leu Pro Val LeuAla Asp Arg Val 305 310 315 320 Ile Ile Pro Phe Ser Gly Trp Cys Asp GlyAsn Ser Asn Met Ser Arg 325 330 335 Glu Val Val Asp Pro Glu Val Phe PheAsn Ala Thr Gly Cys Leu Arg 340 345 350 Asn Leu Ser Ser Ala Asp Ala GlyArg Gln Thr Met Arg Asn Tyr Ser 355 360 365 Gly Leu Ile Asp Ser Leu MetAla Tyr Val Gln Met Cys Val Ala Ala 370 375 380 Ser Arg Cys Asp Asp LysSer Val Glu Asn Cys Met Cys Val Leu His 385 390 395 400 Asn Leu Ser TyrArg Leu Asp Ala Glu Val Pro Thr Arg Tyr Arg Gln 405 410 415 Leu Glu TyrAsn Ala Arg Asn Ala Tyr Thr Glu Lys Ser Ser Thr Gly 420 425 430 Cys GluSer Asn Lys Ser Asp Lys Met Met Asn Asn Asn Tyr Asp Cys 435 440 445 ProLeu Pro Glu Glu Glu Ile Asn Pro Lys Gly Ser Gly Trp Leu Tyr 450 455 460His Ser Asp Ala Ile Arg Thr Tyr Leu Asn Leu Met Gly Lys Ser Lys 465 470475 480 Lys Asp Ala Thr Leu Glu Ala Cys Ala Gly Ala Leu Gln Asn Thr Thr485 490 495 Ala Ser Lys Gly Leu Met Ser Ser Gly Met Ser Gln Leu Ile GlyLeu 500 505 510 Lys Glu Lys Gly Leu Pro Gln Ile Ala Arg Leu Leu Gln SerGly Asn 515 520 525 Ser Asp Val Val Arg Ser Gly Ala Ser Leu Leu Ser AsnMet Ser Lys 530 535 540 Lys Pro Leu Leu Met Lys Val Met Gly Asn Gln ValPhe Pro Glu Val 545 550 555 560 Thr Arg Leu Leu Thr Ser His Thr Gly AsnThr Ser Asn Ser Glu Asp 565 570 575 Ile Leu Ser Ser Ala Cys Tyr Thr ValArg Asn Leu Met Ala Ser Gln 580 585 590 Pro Gln Leu Ala Lys Gln Tyr PheSer Ser Ser Met Leu Asn Asn Ile 595 600 605 Ile Asn Leu Cys Arg Ser SerAla Ser Pro Lys Ala Ala Glu Ala Ala 610 615 620 Arg Leu Leu Leu Ser AspMet Trp Ser Ser Lys Glu Leu Gln Gly Val 625 630 635 640 Leu Arg Gln GlnGly Phe Asp Arg Asn Met Leu Gly Thr Leu Ala Gly 645 650 655 Ala Asn SerLeu Arg Asn Phe Thr Ser Arg 660 665 15 amino acids amino acid linearpeptide internal 12 Gly Asn Ile Lys Ser Tyr Phe Arg Lys Leu Asn Glu SerGln Val 1 5 10 15 14 amino acids amino acid linear peptide 13 Tyr GluThr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val 1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule, wherein saidmolecule comprises a nucleotide sequence encoding a polypeptide having asequence that is at least 85% identical to the sequence of the humanALARM polypeptide shown in FIG. 1 (SEQ ID NO:2).
 2. The nucleic acidmolecule of claim 1, wherein said nucleotide sequence encodes apolypeptide that binds presenilin-1.
 3. The nucleic acid molecule ofclaim wherein said molecule encode s the polypeptide shown in FIG. 1(SEQ ID NO:2).
 4. The isolated nucleic acid molecule of claim 1, saidmolecule comprising the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1).
 5. An isolated nucleic acid molecule encoding an ALARMpolyieptide, said molecule hybridizing to the complement of the nucleicacid sequence shown in FIG. 1 (SEQ ID NO:1 ) at 50° C. in Church Buffer(7% SDS, 0.5% NaHPO₄, 1 mM EDTA, 1%BSA) and washing at 50C. in 2×SSC. 6.A vector comprising the recombinant nucleic acid of claim
 1. 7. A cellcomprising the recombinant nucleic acid of claim
 1. 8. The nucleic acidmolecule of claim 5, wherein said molecule encodes the polypeptide shownin FIG. 1 (SEQ ID NO:2).
 9. The isolated nucleic acid molecule of claim5, said molecule comprising the nucleotide sequence shown in FIG. 1 (SEQID NO:1).
 10. A vector comprising the recombinant nucleic acid of claim5.
 11. A cell comprising the recombinant nucleic acid of claim 5.